The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be or to describe prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation through kinases of proteins, which enables regulation of the activity of mature proteins by altering their structure and function.
The best characterized protein kinases in eukaryotes phosphorylate proteins on the hydroxyl substituent of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylating serines and threonines, and those specific for phosphorylating tyrosines. Some kinases, referred to as “dual specificity” kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues.
Many kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway.
Non-receptor tyrosine kinases may be recruited to the plasma membrane where they mediate cellular signaling by cell surface receptors lacking intrinsic protein tyrosine kinase activities. For instance, members of the Src family of protein tyrosine kinases are activated in response to stimulation of growth factor receptors and G-protein coupled receptors, as well as many other extracellular stimuli (Thomas, er al., 1997. Annu. Rev. Cell. Dev. Biol. 13:513–609).
Src family kinases have been found associated with coated membrane regions in platelets (Stenberg, et al., 1997. Blood 89:2384–93). Src copurifies with synaptic vesicles in PC12 cells (Linstedt, et al., 1992. J. Cell Biol. 117:1077–84). Src associates with and phosphorylates several proteins involved in membrane trafficking, such as the neuronal synaptic vesicle associated protein synapsin I, synaptophysin and synaptogyrin (Barnekow, et al., 1990. Oncogene 5:1019–24; Foster-Barber, et al., 1998. Proc. Natl. Acad. Sci. USA 95:4673–7; Janz, et al., 1998. J. Biol. Chem. 273:2851–7).
Small GTPases represent a large family of proteins that act as molecular switches that control diverse biological functions, including cell proliferation and differentiation, cytoskeletal organization, protein transport, cell cycle and free radical production (Bourne, H. R. et. al., Nature, 348(6297):125–32, 1990. Bourne, H. R. et. al., Nature, 349(6305):117–27, 1991). That GTPases modulate these central cellular pathways suggest that both GTPases and their regulators are of critical importance.
The dbl homology region (DH domain) is a region of approximately 250 amino acids initially found in the Dbl and Cdc24 proteins. Many proteins have been found to contain DH domains. Many DH containing proteins exhibit cellular transformation activity upon N-terminal truncation. (Reviewed in Quilliam, L. A., et. al. BioEssays, 17:395–404, 1995. Whitehead, I. P., et. al., Biochemica et Biophysica Acta, 1332:F1–F23, 1997. Cerione, R. A. and Zheng, Y. Curr. Opin. Cell. Biol. 8:216–222, 1996).
The DH/PH modules of many Dbl family proteins have been shown to regulate the activity of various Rho sub-family small GTPases by serving as Guanine Exchange Factors (GEF) (Whitehead, I. P., et. al., Biochimica et Biophysica Acta, 1332:F1–F23, 1997. Cerione, R. A. and Zheng, Y. Curr. Opin. Cell Biol. 8:216–222, 1996). Many Rho family GTPases have been shown to regulate the assembly of actin structures and may also regulate gene transcription through various kinase pathways (Hall, A. Science, 279:509–514, 1998).